Oxygenation of steroids



Patented Nov. 3, 1953 UNITED STATES PATENT OFFICE OXYGENATION 0F STEROIDS Delaware No Drawing. Application April 21, 1953, Serial No. 350,218

13 Claims.

This invention is concerned with a method for the oxygenation of certain steroid compounds by means of selected cultures of microorganisms. In particular, it is concerned with the introduction of an hydroxyl group at the ll-position of the steroid nucleus in the ii-configuration. A particularly useful reaction which may be accomplished with the present process is the conversion of compound S (Reichsteins Substance S or 17- hydroxy-ll-desoxycorticosterone) to compound F (Kendall's Compound or 17-hydroxycorticosterone).

The present application is a continuation-inpart of the earlier filed application Serial No.

322,578, filed on November 25, 1952 by Gilbert M.

Shull et al.

The preparation of biologically active steroid compounds, such as cortisone and compound F, is fraught with many great difiiculties. One of the most difficult problems is the introduction of oxygen atoms at essential positions in the steroid nucleus, particularly at the ll-position of this nucleus. Compound S is available by known synthetic routes from various naturally occurring,

relatively cheap, steroid starting materials, such as the vegetable-type steroid compounds. Compound F, on the other hand, is considerably more difiicult to obtain and is a very valuable compound, particularly useful in the treatment of rheumatoid arthritis and certain other conditions of the human body. Any process whereby compound S may be converted to compound F in good yield and without undue expense is of tremendous value to the pharmaceutical industry and to the public in general.

Methods have previously been reported for converting compound S to compound F by means of organisms entirely different than those described below for use in the present process. In U. S. Patent 2,602,769 there is described the use of Cunninghamella blakesleena (of the order Mucorales) and in a publication appearing in the Journal of the American Chemical Society (vol. 74, p. 2381 (1952) a process utilizing Streptomyces fradz'ae is described. A great number of microorganisms has been tested for their effectiveness in converting compound S to compound F. Most of these gave no indication of such 11- fl-hydroxylation. A considerable number of cultures of organisms of the order Mucorales have been tested for the same reaction, few of these showed even a low order of conversion. In contrast to the published methods and the poor results obtained with a variety of organisms studied by the inventors of the present process microorganisms of the genus Curvularia have given strikingly favorable results, allowing the preparation of such products as compaund F in yields of up to 40% or more. Furthermore, the reactions can be conducted under such conditions that the products may be isolated with relative ease and in high purity. This makes possible the practical preparation on a large scale by biochemical methods of such substances as compound F for the first time.

It has been found that by contacting a steroid compound, in particular those having a methylene group at the ll-position, with the oxygenating activity of certain selected microorganisms, i. e. with the organisms themselves or with enzyme systems of the organisms, the selective 11- B-hydroxylation of these steroid compounds may be accomplished. Among other reactions which may be accomplished is the conversion of compound S to compound F. The organisms with which we are concerned are oxygenating strains of fungi from the genus Curvularia, which genus belongs to the order Moniliales of the class Fungi Imperfecti. This is based on the use of the classification of Saccardo, P. A., Sylloge, Fungorum, vol. 8. Of particular value are strains of the species Curvularia falcata, C. brachyspora, C. Zunata, C. pallescens and other species of this genus. Other microorganisms from the genus Curvularia may be selected for conducting the process of this invention by simple tests which will be described in more detail below. Many of these organisms are available in public culture collections and others may be isolated from the natural materials, such as soil, by standard procedures well known to mycologists.

As indicated above, the process of the present invention may be used for the conversion of compound S to compound F. However, the process may also be used for the lbs-oxygenation of a variety of other steroid compounds which are unsubstituted in the ll-position of the nucleus. Various side chains may be present at the 17-position of the nucleus and keto or hydroxyl groups may be present at the 3-position of the nucleus. The steroid compounds used as substrates for the reaction may also bear carbon to carbon double bonds at various points of the nucleus, such as the 3,4- or 5,6-position. It should be realized that yield or oxygenated product will vary to some extent with the nature of the steroid compound used as starting material, with the particular strain of Curvularia used, and with the conditions employed for the reaction (i. e. temperature, time, pH, nutrient medium, time at which the comcific steroid compound, whereas a second steroid compound used under otherwise exactly identical conditions may give only a moderate orpooryield of the desired compound. Thepresence of an hydroxyl group at the ZI-position of the pregnene-type steroid structure may be particularly useful in assuring a good yield of the I'1-';9-'hydroxy compound. Since the cortical steroid type compounds possess such an hydroxyl group, the present process is particularly useful for preparing the ll-B-hydroxylated compounds of this series. Among the products which have been converted to the corresponding ll-e-hydroxylated products are compound S anddesoxycorticosterone. Various methods may be used in the evaluation of the products produced by these processes For instance, if a steroid compound with a suitable side chain is used, the proportion of the product produced may be evaluated by determination of the effect on adren'alectomlzed mice or upon the eosinophil count of experimental animals. Furthermore, the pure products produced by the hydroxylation reaction may be isolated as described below.

The efiectiveness of a chosen microorganism for the process of this invention may be determined by cultivating the organism in a suitable nutrient medium containing carbohydrates, salts, sources of organic nitrogen and so forth. The steroid compound as a solid or as solution in a 'suitable'solvent, for example, acetone or ethanol, is added to the cultivated microorganism under sterile conditions and the mixture is agitated and aerated in order to bring about the growth of the microorganism and oxygenation of the steroid substrate. The steroid may be added when the medium isseeded under sterile conditions witha culture of the microorganism or after growth of the organism is established. In some cases it may be found advisable to add the steroid compound after growth of the microorganism has been established in the nutrient medium under aerobic conditions. This is particularly true if, during the initial stages of growth of the microorganism, there is a tendency to produce u'ndesired by-products from the steroid substrate. The acetate or other ester of a steroid maybe used in place of the alcohol itself, although this may sometimes lead to an appreciably lowered yield of hydroxylated product. Alternatively, enzyme preparations from the growth of "a suitable oxygenating organism of the genus Curvularia may be used for conducting the process. A further, most useful method is one in which the microorganism is grown on a suitable nutrient medium under aerobic conditions in the absence of the steroid. The mycelial growth may then be filtered from the broth and may, if desired, be washed with distilled water. The mycelium is then suspended in distilled water containing the steroid substrate. Agitation of the mixture and aeration is continued for a period of fromabout .12 to 48 hours after which the products of the reaction are recovered. This process has the advantage of ease of recovery of the steroid compound, since the various nutrient material originally used to obtain growth of the microorganism are now absent as well as the various materials excreted by the growing organsims during the initial period. With some strains of the genus Curvularia even better total yields of oxygenated products are obtained by this method than is the case whenthe steroid is added at the beginning or at an intermediate period directed to the whole fermentation broth. Other methods familiar to enzyme chemists may be utilized for conducting the present oxygenating process. The proportion of products and the rate of oxygenation, as well as the nature of the by-products formed, may vary depending on the use of the whole fermentation broth or of the isolated washed mycelium.

In gcner'ala concentration of not greater than one to two percent by weight of the total weight of substrate, for instance, the compound S-type material, is used in conducting this process, although sometimes other concentrations may be found to be more favorably used. Since the solubility of the starting material in water is quite limited, an excess of the material may be slowly converted to the oxygenated product. However, the state of subdivision of the steroid when added to the oxygenating system, i. e. growing microorganism or enzyme system, does not seem "to greatly effect the yield and nature of the products under otherwise identical conditions. If a 'watere miscible solvent solution of the steroid compound is added to the aqueous fermentation system, the steroid is generally precipitated in finely divided form in the presence of a large excess of water. This does not seem to appreciably improvethe rate of reactionas compared to the additiorn of dry, relatively large crystals of the steroid.

After completion of the oxygenation process, the product maybe recovered from the mixture by extraction with a "suitable water immi'scible solvent. Chlorinated lower hydrocarbons, ketones, and alcohols are useful. These include chloroform, methylene chloride, trichlorethane, ethylene dichloride, and so forth. The use of hot ethylene dichloride, i. e. from about 40 to about C is particularly favored for the extraction of the steroid products. The extract of product and unreacted starting material may be concentrated to a small volume or to dryness to obtain a solid product. Purification of the product may be accomplished inseveral ways. I Most useful is the separation by means of chromatography of the product from starting material and from other products such as 'mo're highly oxygenated materials that may beformed during the reaction. Adsorbe nts such as silica gel or other suitable adsorbents are particularly useful for this purpose. It has been fou'ndthat a' column prepared irom a mixture of silica gel and a lower alcohol, especially ethanol, is particularly useful for theseparation of the ste r oid starting, materials. The steroid mixtures may be applied to columns of adsorbents suchas silica gel inconcentrated chloroform or methylene chloride solution. The column may then be washed 'with additional amounts of the solvent to remove such impurities as fats and pigments. The adsorbed mixture then is separated by the gradual addition of a mixture of the solvent together with a small percentage, for example 1 to 5%, of a lower alcohol (methanol, ethanol, etc.). The materials may be separated and the separated compounds gradually eluted from thecolumn by utilization of a mixture of solvents of gradually increasing polarity; for instance, a mixture of methylene chloride and a minor, gradually increasing amount of ethanol is very useful.

Fractions of the eluted material from chromatographic columns may be-checked for the nature of the product by subjecting small portions of the solutions to chromatography on paper. Methods which are particularly useful for conducting this type of separation and analysis are described in detail in U. S. Patent 2,602,769, issued on July 8 1952, to H. C. Murray et al., in the copending patent application Serial No. 276,678, filed March 14, 1952, by Gilbert M. Shull et al., and in a publication by Shull et al. in the Archives of Biochemistry, vol. 37, p. 186 (1952). This method is also very useful for evaluating new strains of microorganisms to determine their usefulness in the process of this invention. The fermentation may be conducted on a small scale with the steroid or compound S or a suitable compound S derivative as the substrate and the whole extract of the fermentation mixture may be concentrated and subjected to paper chromatography. By utilizing known samples of compound S, compound F and other related products for comparison, it is possible to determine whether the chosen microorganism is suitable for the present process. I

Descending paper chromatograms utilizing paper treated with a 35% solution of propylene glycol and developed with a mixture of 78 volumes of toluene and 22 volumes of dioxane may be utilized for the rapid evaluation of various strains of the preferred microorganisms. Such a separation can be completed in as little as three hours and the paper chromatogram, after being dried, may be examined under ultraviolet light in a fluorescent scanner, such as that of Haines and Drake (Federation Proceedings 1950, vol. 9, p. 180), to determine the position of the various materials, such as compounds S and F, by their fluorescence. The zones in which the various substances occur may be marked and cut from the sheet or strip of paper. The material may then be eluted with a solvent such as ethanol and obtained as practically pure solid material by evaporation. A quantitative analysis of such a mixture may be accomplished in this manner. The amount of the isolated products may be determined, for example by measuring the ultraviolet absorption of solutions of the materials. Particularly useful is the absorption characteristics of these compounds when dissolved in concentrated sulfuric acid.

After separation of the reaction products by column chromatography, the desired fractions may be combined and concentrated to a small volume. The product may then be crystallized from a suitable solvent such as ethyl acetate. One product prepared by application of the present process has been compared with samples of authentic compound F and has been found to be identical in all respects. Corticosterone prepared by the process of this invention has also been compared with a standard sample and shown to be identical with it.

The following examples are given by way of illustration and are not intended as a limitation of this invention. Indeed, as many apparently widely different embodiments of the present invention may be made without departing from the spirit and scope hereof, it is to be understood that the invention is only limited as defined in the appended claims.

Example I A culture of an organism received from the culture collection of the Quartermaster Corps 6 at Philadelphia and designated by them as Curvularia ,faleata QMlZOh was propagated on an agar nutrient medium. We have now established that this organism belongs to the C. Zunata species, rather than the C. falcata species, and shall therefore refer to it hereinafter as Curimlaria. Zunata (QM120h). A living culture of this organism has been deposited with the Fermentation Division of the Northern Regional Research Laboratory at Peoria, Illinois, and has been added to its permanent collection of microorganisms as NRRL-2380. The organism was rinsed from the agar slant under sterile conditions into a sterile medium having the following composition Per cent Malt extract 5 Sucrose 1 Sodium nitrate 0.2 Potassium chloride 0.05 Magnesium sulfate heptahydrate 0.05 Ferrous sulfate heptahydrate 0.05

Dipotassium acid phosphate 0.1 Distilled water, adjusted to pH 7.0 with potassium hydroxide.

One hundred milliliters of this medium was used in each of several three hundred-milliliter flasks. To each flask was added 50 milligrams of compound S dissolved in a small volume of acetone. Throughout these operations the fermentation mixture was maintained under sterile conditions. The mixture was then shaken for a period of seven days at a temperature of about 28 C. The contents of the flasks were combined and extracted with several portions of ethylene dichloride using one-fifth the volume of the aqueous phase each time. Thecombined ethylene dichloride extracts were dried over anhydrous sodium sulfate and, after the drying agent was removed, the solvent was removed (one milliliter of solvent per gram of silica gel) The column was developed by means of a mix ture of 97 volumes of methylene chloride and three volumes of ethanol. The efliuent from the column was collected in small fractions of equal volume and periodically these were examined by means of paper chromatography in order to separate the fractions containing the desired product. All of these fractions were combined and concentrated under vacuum to dryness to obtain the solid product. This was proved to be compound F by comparing with a known sample of the same material.

Example I! A culture of Curvularia, Zunata (QMh) was grown in flasks containing the same medium described in Example I. One hundred milliliters or thisinoculum'i was; added under: sterile: come-1 tions to= two. liters 01-1 the; following medium:

This mixture was adjusted to' pH 7 with sulfuric a'cid and: 035% of calcium carbonate was addedbeibre the mixture was' sterli'zecl'. The inoculated medium was aerated at the rate of about one-halt to one volume of air per volume of solhtlonper minute at 27 to 28 C. for 24 hours. During this time the mixture was stirred at the rate of about I700 revolutions'per' minute. Onehalf gram of compound S in the form of the alcohol' was dissolved 2O milliliters of 96% ethanol. The solution was added to theferm'entatibn mixture under sterile conditions. The reaction was then continuedv for a. further 24 hours under exactly the same conditions as described above.

The whole fermentation mixture was removed from. the fermentation. vessel. The mixture was extractedtwice withan equal volume of ethylene dichloride at.'70." C.. The extracts were combined and'evaporated tov dryness. The dry solids were dissolved ina small volume of, methylene chloride and thesolutionwas added to a column of. silica gel. The silica gel column had' been. prepared previously by treating each gram of. silica gel' with one milliliter of 95% ethanol. This mixture was. suspended in methylene chloride and poured into a chromatographic column. After the steroid mixture had. been introduced into the column it was washed with. several portions or". methylene chloride to remove. fats and pigments. The column was then. developed by adding a mixture of 97 volumes of. methylene chloride and" 3 volumes of. ethanol. The eluate was.- divided into a. series of. small fractions. Portions of these were. analyzed bymeans. of the paper chromatographic system described above and those fractions containing the. same compound were combined. It was found that. the first material leaving the column was recovered compound S. I This material is recoverable and may be reused; The second material leaving the column was an unidentified steroid. The third material leaving the column was recovered and shown to be compound F; By" removing the solvent from the combined" fractions containing the compound F, there was obtained in yield of over. 35%- a dry product which may be readily'f'urther purified.

Example III A culture of Curvularia lunata' (QM1'02h): as used in Example I was cultivated on the same mediumdescribed in Example I under aerobic. conditions. The mycelium' from two liters of such a mixture obtained after.- 22 hours of growth was filtered, washed with a small volume ofv distilled water and then sus pended in two. liters of distilled water. One-half gram of compound S was added to the mixture. This preparation was stirred and aerated at the rate of one-half volume of air per volume of mixture per minute for 16 hours. Thea mixture was then extracted with one-half volume" of chloroform; threev times. The combined! chloroform: extracts were; concentraterf to a. smalli volume and the mixture: of steroids wasrpurmed hm column chromatograpliy.- onsilica: gel. A; yieldof approximately 35% of pure compound E was obtained. In addition, approximately 10% of. the compound S used: as starting materialv was recovered inpure form.

Example IV A culture of an organism available: from; the: Philadelphia Laboratories of the Quartermaster. Corps'under the code- No: Curvularia' sp. QM3f7-1-d was grown inflasks under aerobic: conditions on the medium described in Example I. This onganism' has now beendefinitely established. as: belonging to:the (5'. pallescens species, and shall accordingly be' specifically identified hereinaetteras Cumulam'a palleseensr (QMBZ 1d)- A living; culture of this: organism has been: deposited: with;

. the Fermentation Division. of the: I-Iortherrr Re.

gional Research Laboratory at Peoria, Illinois, andhas been added toaits permanent collection: of microorganisms as HEEL-2381. After 24; hours growth 100' milliliters of this mixture was.

. used to seed 2 liters of the medium described: im

Example 11. After the two-liter mixture: had been grown under aerobic conditions for- 2s: hours; one-hale gramof compound S was: added; to. the fermentation mixture under sterile conditions. The mixture-was: stirred at. 28 G:- tor a. further 20 hours, aerating and agitatingthroughs out. The mixture was removed. from' the vessel: in which the fermentationhad been: conducted-l and was extracted three times Wit-hi one-haltf volume of chloroform eaclr time; The extracts: were: combined and concentrated? to a. smalh vol!- ume. The mixture-was then: placed: cm a columm of. silica gel which had been prepared; as. des scribed above. The silica gels columm was; washed with methylene chloride and then de velopedawith a solution of ethanolain: methylene chloride. From thecolumn was recovered some: compound 8 starting material as. well as; the. llrsroxygenated product compound E. The: isolated. product was purified and its melting:

I point and other physicalproperties were sourpared to that of a pure synthetic: sample. These: were shown tov be identical. Furthermore. the; compoundF prepared: in. this manner was tested for. its. eflect in the mouse: glycogen: storage; fatin animals which had. been. adrenalectonnzsdr Itproved to be. highly active; further establishing the nature of the product.

Ii'xam'ple V" A. culture. of (luroularia. lunata. (QMLZOh) was grown as described in Example Iaboves. except. that desoxycorticosterone was: used in P1926 of compound S.. After the conclusion of the termentation, the: crude product was, extracted by means of methyl isobutyl: ketone. and. the solvent. was then removed. under vacuum. The residual product was tested for its activity in liver glazed-- gen storage in adrenalectomized mice and. the positive result showed. the introduction of, an. ll-p-hydroxyl in the steroid. Purification by column chromatography as described. above. gave. approximately 28% yield of a crystalline compound that was shown to be corticosterone by its physical constants.

What is claimed is:

1. A process for the ll-fl hydroxylation of a steroid compound, which comprises contacting said steroid compound with the oxygenating activityof an organism chosen from the genus Curvularia.

2. A process as claimed in claim 1 wherein the steroid compound is subjected to the action of a growing culture of the microorganism.

3. A process as claimed in claim 1 wherein the microorganism is cultivated aerobically in a nutrient medium until substantial growth is obtained, the mycelium is separated from the broth and the compound is contacted with an aqueous suspension of the mycelium.

4. A process for the ll-p-hydroxylation of a steroid compound, which comprises contacting said steroid compound with the oxygenating activity of an organism chosen from the group consisting of Curvularia lunata (QM120h) and Curvularia pallescens (QM371d).

5. A process as claimed in claim 4 wherein the steroid compound is selected from the group consisting of compound S and desoxycorticosterone.

6. A process for the conversion of compound S to compound F, which comprises contacting compound S with the oxygenating activity of an organism chosen from the genus Curvularia.

7. A process for the preparation of compound F, which comprises cultivating Curvularia lunata (OM120h) in an aqueous nutrient medium under aerobic conditions in the presence of compound S.

8. A process for the preparation of corticosterone, which comprises contacting desoxycorticosterone with the oxygenating activity of an organism chosen from the genus Curvularia.

9. A process as claimed in claim 1 wherein the organism is chosen from the species Curvularia lunata.

10. A process for the li-p-hydroxylation of a cortical steroid type compound, which comprises Curvularia lunata in an aqueous nutrient medium under aerobic conditions in the presence of said cortical steroid type compound.

11. A process as claimed in claim 6 wherein the organism is chosen from the species Curvularia lunata.

12. A process for the 11-c-hydroxylation of a cortical steroid type compound, which comprises contacting said cortical steroid type compound with the oxygenating activity of an organism chosen from the species Curvularia pallescens.

13. A process for the preparation of compound F, which comprises cultivating an organism chosen from the species Curvularia Zunata in an aqueous nutrient medium under aerobic condi tions until substantial growth is obtained, then separating the mycelium from the broth, and thereafter contacting compound S with an aqueous suspension of such mycelium.

GILBERT M. SHULL. DONALD A. KITA. JACOB W. DAVISSON.

References Cited in the file of this patent UNITED STATES PATENTS Number Name Date 2,602,769 Murray et a1 July 8, 1952 FOREIGN PATENTS Number Country Date 3,476/51 Australia Aug. 30, 1951 4,460/51 Australia Oct. 18, 1951 

1. A PROCESS FOR THE 11-B-HYDROXYLATION OF A STEROID COMPOUND, WHICH COMPRISES CONTACTING SAID STEROID COMPOUND WITH THE OXYGENATING ACTIVITY OF AN ORGANISM CHOSEN FROM THE GENUS CURVULARIA. 